CN102520179A - Quantitative detection method of fumonisins B1 - Google Patents
Quantitative detection method of fumonisins B1 Download PDFInfo
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- CN102520179A CN102520179A CN2011104031787A CN201110403178A CN102520179A CN 102520179 A CN102520179 A CN 102520179A CN 2011104031787 A CN2011104031787 A CN 2011104031787A CN 201110403178 A CN201110403178 A CN 201110403178A CN 102520179 A CN102520179 A CN 102520179A
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Abstract
The invention relates to a quantitative detection method of fumonisins B1, comprising the following steps of: 1, preparing conjugates FB1-KLH and FB1-OVA; 2, preparing anti-FB1 monoclonal antibody; 3, preparing colloid gold, labeling the anti-FB1 monoclonal antibody and spraying the labeled monoclonal antibody onto a gold labeled gasket; 4, spotting on a nitrocellulose membrane; 5, assembling to form strips; and 6, drawing a standard curve of FB1 concentration and color value and dragging the color value of a sample to be tested into the standard curve to obtain the FB1 content in the sample to be tested. The invention has advantages of single detection object and strong pertinence, high accuracy, fast detection speed and short required time. The method provided by the invention can be used for detection without trained professionals. The method satisfies the demand of rapid and correct judgment of FB1 content in food storage and sale institution, exit-entry and custom inspection departments, and is convenient for popularization and application at grass-root level.
Description
Technical field
The present invention relates to a kind of method of technical field of biological, specifically relate to the method for a kind of detection by quantitative fumonisin B1.
Background technology
(Fumonisin B1 is the fusarium moniliforme secondary metabolite that breeding is produced under certain humidity and temperature conditions FB1) to fumonisin B1, in worldwide, distributes extensively.1988, Gelderblom etc. isolated fumonisin first from the fusarium moniliforme nutrient solution.Fumonisin can pollute corn and goods thereof, and in some products that with cereal are raw material like noodles, beer, flavouring, even in asparagus, also detected fumonisin.It is reported, fumonisin and horse white matter of brain malacosis, the pulmonary edema syndrome of pig, diseases such as rat liver cancer are relevant.Except that above-mentioned disease, find that in the area, Transkei and the area studies of Chinese Lin County in South Africa the corn product that edible fumonisin pollutes is relevant with human cancer of the esophagus.The toxin that fusarium moniliforme produces by international cancer research association (International Agency for Research on Cancer IARC) is divided into the 2B class--possible human carcinogenic substance.After the accession to WTO; The quantum of international trade of agricultural product and relevant food increases day by day; Also increasingly high to the requirement of the biological safety of importing and exporting product thereupon; For the smooth listing that guarantees this series products and eater's health, departments such as entry and exit quarantine, customs, manufacturing enterprise, superintendent office press for a kind of special, quick, easy fumonisin detection method.
Colloidal gold-labeled method be with collaurum as the tracer label thing, use a kind of immunolabelling technique of antigen-antibody reaction.Collaurum is by gold chloride (HAuCl
4) under effects such as reductive agent such as white phosphorus, tannic acid/sodium citrate and trisodium citrate, aggregate into the gold grain of specific size, because electrostatic interaction becomes a kind of stable colloidal state, so be called collaurum.Colloid gold label comes down to the process that encapsulates that protein high molecular is adsorbed to the colloid gold particle surface.Adsorption mechanism is the negative charge on colloid gold particle surface, forms strong bonded with the positive charge group of protein molecule because of electrostatic interaction.The colloid gold particle of this ball-type has high electron density; Can be to multiple boiomacromolecule material such as staphylococcus, immunoglobulin (Ig), toxin, glycoprotein, enzyme, microbiotic, hormone, BSA non-covalent combinations such as (bovine serum albumin(BSA)s); Form visible aubergine; Make it become immunoreactive good label, therefore be widely used in the detection of various materials.
Enzyme-linked immunosorbent assay since the antigen (or antibody) in the liquid phase need through diffusion could with antigen or the antibody response on the solid phase, need the long period, each step needs washing up hill and dale, and needs specific enzyme and substrate colour developing, so length consuming time, program is loaded down with trivial details; High performance liquid chromatography is unfavorable for basic unit's conventional sense use because of its requirement to test sample, instrument and operating personnel.Colloidal gold immunity chromatography can overcome the deficiency of said method; Colloidal gold immunochromatographimethod quantitative test method is utilized the antigen-antibody reaction principle, and the colloid gold label tracer is trapped and develops the color with being fixed on antigen or antibody complex formation on the film, does not need specific enzyme and substrate to develop the color; Do not need the physisorption of the long period between the antigen-antibody yet; And judge the yin and yang attribute result and calculate concrete concentration according to typical curve according to the colour developing depth, safe and effective, simple and convenient.
The basis of colloidal gold immune chromatography experiment is the immobilization of antigen or antibody and the colloid gold label of antigen or antibody.The antigen or the antibody that are fixed on the NC film (nitrocellulose filter) still keep its immunologic competence, and the antigen of colloid gold label or antibody had both kept its immunologic competence, and the function of spike is arranged again.When measuring, reacted through capillary action is divided a word with a hyphen at the end of a line forward and gold is marked on the pad antigen or antibody by sample article (measuring wherein antibody or antigen).Continue to divide a word with a hyphen at the end of a line forward; Be fixed on T line (detection line) antigen or antibodies and form immune complex and be trapped and develop the color; Be about one and widely be the brownish red band of 1mm; Unnecessary golden labeling antibody continues to move forward, and closes with two resistive connections that are fixed on C line (nature controlling line) to be trapped and to develop the color, and is about one and widely is the brownish red band of 1mm.The amount that this moment, the T line formed examined object matter in gold mark compound and the sample is certain ratio, so can carry out quantitative test according to the shade that the T line appears.
Several different methods has been set up in detection about fumonisin B1 both at home and abroad.The method that detects fumonisin B1 at present is mainly high performance liquid chromatography (HPLC), and in the corn in the world and the investigation of goods thereof, what the laboratory more than 90% was used all is the HPLC method.Yet; Because fumonisin B1 itself had not both had special ultraviolet absorption group; There is not simultaneously fluorescent characteristic yet; But fumonisin B1 can form the derivant with fluorescence with some substance reaction under certain condition, so the accuracy and the sensitivity of the selection of fluorescence derivating agent and deriving method and HPLC detection fumonisin have substantial connection.This method need be carried out strict pre-service to test sample in addition, also needs expensive instruments such as high performance liquid chromatograph, requires the professional to operate simultaneously, is unfavorable for on-the-spot conventional sense use.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, provide a kind of fast quantification to detect the method for fumonisin B1.Detected object of the present invention is single and with strong points; Accuracy rate is high, and detection speed is fast, and required time is short; Only need 20 minutes; Do not need just can use the inventive method to detect through the professional of training, satisfy grain store inspection departments such as marketing organization, entry and exit, customs fast, the requirement of the FB1 content that judges rightly, and be convenient to basic unit and promote and use.
The present invention realizes through following technical scheme:
A kind of method that detects fumonisin B1, this method comprises the steps:
Step 1, with the FB1 haptens respectively with KLH and OVA coupling, obtain FB1-KLH and FB1-OVA;
Step 6 according to the colour developing value of known variable concentrations FB1 standard solution, is drawn the typical curve of FB1 concentration and colour developing value;
Step 7 is got testing sample and used methanol extraction, and is centrifugal, gets supernatant, and uses diluted, according to the colour developing value of the methanol extract liquid of this testing sample, obtains the content of FB1 in this testing sample extract through typical curve.
Preferably, in the said step 3, the particle diameter of said collaurum is 25nm.
Preferably, in the said step 3, the preparation of said collaurum comprises the steps: the 0.01%HAuCl with 100ml
4Solution is heated to boiling, adds 1% citric acid three sodium solution of 1ml afterwards, boils 7～10min, adds tri-distilled water at last to 100ml, prepares the colloidal gold solution of 25nm, and said percentage is mass and size percentage.
Preferably, in the said step 3, the monoclonal antibody of said anti-FB1 was handled through the dialysis desalination before being labeled.
Preferably, in the said step 3, said mark comprises the steps: under stirring condition, in colloidal gold solution, adds the monoclonal antibody of anti-FB1, makes its final concentration reach 7.2 μ g/mL, hatches 15min under the room temperature, uses 0.1mol/L K
2CO
3The pH that regulates colloidal gold solution is 6.5, and the final concentration that adds 10%BSA to BSA then is 0.1%, the centrifugal 20min of 10000rpm; Abandon supernatant; Using the 2mM pH contain 1%BSA again is 8.0 borate buffer solution washing, washs altogether 3 times, adds at last to contain 4% sucrose, 6% trehalose, BSA and Sodium azide to be respectively 1% and 0.05% 2mM pH be in 7.4 the borate buffer solution; Promptly accomplish mark, said percentage is mass and size percentage.
Preferably, in the said step 5, said drying is 37 ℃ of oven dryings.
Preferably, in the said step 7, said methyl alcohol is 80% methyl alcohol, and said percentage is percent by volume.
Preferably, in the said step 7, said centrifugal be centrifugal 15 minutes of 2500g.
Preferably, in the said step 7, said dilution is that the 0.05M pH that contains 10% methyl alcohol is 7.4 PBST, and said percentage is percent by volume.
Compared with prior art; The present invention has following beneficial effect: testing sample is if there is the FB1 toxin; Because capillary effect chromatography forward moves; FB1 in the sample liquid and the compound that gold mark monoclonal antibody forms have competed the chance that antigen combines with gold mark monoclonal antibody on the detection line, thus collaurum can not or the seized survey line of only a small amount of ability on antigen dam and deposit; So judge FB1 content with the detection line colored intensity, but and accurate quantification judge the content of FB1 in the test sample such as cereal, food, animal product.Detected object of the present invention is single and with strong points; Accuracy rate is high; Detection speed is fast, and required time is short, only need 20 minutes, do not need just can use the inventive method to detect through the professional of training; Satisfy grain store inspection departments such as marketing organization, entry and exit, customs fast, the requirement of the FB1 content that judges rightly, and be convenient to that basic unit promotes and utilization.
Description of drawings
Fig. 1 is the structural representation of embodiment of the invention test strips;
Fig. 2 is the synoptic diagram as a result of embodiment of the invention test strips.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Among the following embodiment, the experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.Among the present invention, BSA is a bovine serum albumin(BSA); OVA is an oralbumin; FB1 is fumonisin B1; KLH is a keyhole limpet hemocyanin; FB1-KLH, FB1-OVA represent the conjugate of FB1 and KLH, OVA respectively, and the PBST representative adds the damping fluid that the Twen-20 configuration forms in PBS, and these technical terms all are well-known to those skilled in the art.
Embodiment
1, the preparation of antigen
(1) preparation of fumonisin B1 immunizing antigen
1ml KLH (10mg/ml) is put into bag filter, place 200ml to contain 4 ℃ of dialysis of PBS solution 16h of 0.2% (V/V) glutaraldehyde (GA), change the 8h that dialyses among the PBS then over to and remove unreacted GA.In the KLH dialysate, add 1mgFB1,4 ℃ of reaction 16h.Add 10mg Tris, reaction 2h seals unreacted protein loci, and with PBS dialysis 2～3d ,-20 ℃ of preservations obtain the FB1-KLH comlete antigen at last.
(2) preparation of fumonisin B1 envelope antigen
2.5mg oralbumin (OVA) is dissolved in the 0.1ml 0.01M PBS damping fluid, adds 10 μ l 50% (V/V) GA, stirred overnight at room temperature.Use the PBS dialysed overnight under 4 ℃ of conditions, remove unnecessary GA.Get 0.5mg fumonisin (FB1) and be dissolved in 0.2ml25% (V/V) ethanol, it is joined in the OVA dialysate (about 0.15ml) of activation, add 0.1ml 1M carbonic acid buffer (pH9.5), 4 ℃ of stirred overnight.Add 0.05ml1M lysine (pH7), 4 ℃ of reaction 3h.At last with the PBS 72h that dialyses, change liquid 2 times ,-20 ℃ of preservations prepare the FB1-OVA comlete antigen.
2, fumonisin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR
FB1-KLH comlete antigen freeze-dried powder is dissolved among the PBS, measures the concentration of carrier protein in the comlete antigen.The Freund's complete adjuvant of antigen and equivalent is fully emulsified, hypodermic injection immunity Balb/C mouse in 6 age in week, every 0.1ml; Two exempt from: after two weeks, use incomplete Freund instead, use the same method and dosage, carry out immunity; Three exempt from, and operation is exempted from two.Immunity after 5 days eyeground vein blood sampling survey and tire, booster immunization when above that reaches 1: 10000 of tiring: the antigen 0.1ml that lumbar injection does not add adjuvant, put to death mouse after three days, get its spleen, merge with the myeloma cell.Screen positive hybridoma cell with indirect ELISA method.Through a large amount of preparation of mouse peritoneal injection hybridoma mouse ascites, behind the filtration of ascites process, the centrifugal preliminary purification, adopt sad method and affinity chromatography purifying ascites, prepare the monoclonal antibody of anti-fumonisin B1.
3, the preparation of collaurum
Elder generation is with 0.01% (W/V) HAuCl of 100ml
4Solution is heated to boiling, adds 1% (W/V) trisodium citrate aqueous solution of 1ml rapidly, begins some blueness; Light blue then, blue, redness appears in heating again, boils 7～10min and transparent claret occurs; Add tri-distilled water at last to 100ml, promptly prepare the colloidal gold solution of 25nm.Use the Electronic Speculum microscopy then, guarantee that the gold grain for preparing makes its size consistent as far as possible, evenly, particle diameter is about 25nm.
4, the mark of monoclonal antibody
Antibody protein to be marked is with 48 hours desalinations of sodium chloride solution dialysis of 0.005mol/L, and the collaurum with the 25nm for preparing comes mark polyclonal antibody albumen then.Concrete steps are: 1. add antibody protein in the colloidal gold solution in stirring rapidly and make its final concentration reach 7.2 μ g/mL, hatch 15min under 25 ℃ of room temperatures; 2. use 0.1mol/L K
2CO
3The pH that regulates gold solution is 7.0, adds 10% (W/V) BSA then and comes the stable colloid gold solution to final concentration 0.1%, reaction 5min; 3. the centrifugal 20min of 10000rpm abandons supernatant, is 8.0 borate buffer solution washing again with the 2mM pH that contains 1% (W/V) BSA, washs altogether 3 times; 4. contain in the 2mM borate buffer solution (pH 7.4) that 4% (W/V) sucrose, 6% (W/V) trehalose, BSA and Sodium azide be respectively 1% (W/V) and 0.05% (W/V) the last adding, and 4 ℃ store for future use.More than it should be noted that in the operation should impure particulate, available high speed centrifugation or miillpore filter pre-service in all solution.
5, the assembling of colloidal gold strip
The monoclonal antibody of mark is sprayed on the gold mark pad; FB1-OVA is sprayed to the T line on the nitrocellulose filter; The monoclonal antibody of the anti-mouse of rabbit is sprayed to the C line on the nitrocellulose filter, afterwards nitrocellulose filter is put into bovine serum albumin solution and seal; With gold mark pad, the nitrocellulose filter after the spraying, sample pad and adsorptive pads are assembled into test strips, 37 ℃ of oven dryings.The assembling sequence of test strips is as shown in Figure 1; The composition of test strips comprises plastics end liner 1, nitrocellulose filter 2, gold mark pad 3, sample pad 4, adsorptive pads 5 from down to up successively; Wherein the effect of plastics end liner 1 provides assembly platform, has the C line with the anti-mouse monoclonal antibody spraying of rabbit on the nitrocellulose filter 2, with the T line of FB1-OVA spraying; Be added with golden labeling antibody on the gold mark pad 3, the position that sample pad 4 provides testing sample to add.
6, the use of colloidal gold strip and result judge
Get the FB1 standard items, respectively dilution be 10,20,40,80,160,320ng/ml solution, and prepare blank solution simultaneously, get 250 μ l and splash in the test strips sample cell; Get 0.75g sample to be checked with 3ml80% methyl alcohol (methyl alcohol: water=80: 20) extract; The centrifugal 15min of 2500g gets supernatant, and with 2.4 times of diluted; Getting 250 μ l splashes in the test strips sample cell; The test strips of the standard items accomplished and testing sample of will developing the color behind the 20min is put into and is read the bar appearance, reads the colour developing value of C, T line, and record; Utilize Excel software to draw the typical curve of FB1 standard items concentration and colour developing value, testing sample colour developing value is brought in the typical curve, obtain the FB1 concentration results; With this result * dilution gfactor (4 * 2.4), be contained FB1 concentration (μ g/kg) in the sample.
Splash into sample to be checked in the sample slot of test strips; Because capillary effect; The chromatography direction of liquid upwards; Result for chromatography sees Fig. 2, and wherein a is the synoptic diagram as a result when FB1 content surpasses 320ng/ml in the sample, b1-b4 be contain that FB1 concentration is respectively 10,40,80, the synoptic diagram as a result during 320ng/ml.
Authentication method is specially:
If on the C line, red stripes occurs, judge that then the test strips test result is effective;
If on T line and C line, the brownish red band all occurs, in T line colour developing value substitution typical curve;
If T line colour developing value is equal to or greater than the value of blank solution, then do not contain or contain the FB1 that is less than 10ng/ml in the sample;
If T line colour developing value is in standard curve range, the FB1 concentration that then contains in the sample can be calculated according to typical curve;
If T line colour developing value is 0, then FB1 content exceeds 320ng/ml in the testing sample.
Can find out; But the FB1 in the method direct quantitative test sample of present embodiment; Do not need professional training, easy to operate, quick, can obtain the result in 20 minutes; The present invention satisfy grain store inspection departments such as marketing organization, entry and exit, customs fast, the requirement of the FB1 content that judges rightly, and be convenient to that basic unit promotes and utilization.
Claims (9)
1. a method that detects fumonisin B1 is characterized in that, comprises the steps:
Step 1, with the FB1 haptens respectively with KLH and OVA coupling, obtain FB1-KLH and FB1-OVA;
Step 2 as immunogene, adopts conventional method to prepare the monoclonal antibody of anti-FB1 with said FB1-KLH;
Step 3, the preparation collaurum with the monoclonal antibody of the said anti-FB1 of this colloid gold label, sprays to the monoclonal antibody behind the mark on the gold mark pad;
Step 4 is carried out the spraying of FB1-OVA, rabbit anti-mouse antibody on nitrocellulose filter, afterwards the nitrocellulose filter after the said spraying is put into bovine serum albumin solution and seal;
Step 5, with said gold mark pad, the nitrocellulose filter after the said spraying, sample pad and adsorptive pads are assembled into test strips, drying;
Step 6 according to the colour developing value of known variable concentrations FB1 standard solution, is drawn the typical curve of FB1 concentration and colour developing value;
Step 7 is got testing sample and used methanol extraction, and is centrifugal, gets supernatant, and uses diluted, according to the colour developing value of the methanol extract liquid of this testing sample, obtains the content of FB1 in this testing sample extract through typical curve.
2. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 3, the particle diameter of said collaurum is 25nm.
3. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 3, the preparation of said collaurum comprises the steps: the 0.01%HAuCl with 100ml
4Solution is heated to boiling, adds 1% citric acid three sodium solution of 1ml afterwards, boils 7～10min, adds tri-distilled water at last to 100ml, prepares the colloidal gold solution of 25nm, and said percentage is mass and size percentage.
4. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 3, the monoclonal antibody of said anti-FB1 was handled through the dialysis desalination before being labeled.
5. the method for detection fumonisin B1 as claimed in claim 1; It is characterized in that in the said step 3, said mark comprises the steps: under stirring condition; The monoclonal antibody that in colloidal gold solution, adds anti-FB1; Make its final concentration reach 7.2 μ g/mL, hatch 15min under the room temperature, use 0.1mol/L K
2CO
3The pH that regulates colloidal gold solution is 6.5, and the final concentration that adds 10%BSA to BSA then is 0.1%, the centrifugal 20min of 10000rpm; Abandon supernatant; Using the 2mM pH contain 1%BSA again is 8.0 borate buffer solution washing, washs altogether 3 times, adds at last to contain 4% sucrose, 6% trehalose, BSA and Sodium azide to be respectively 1% and 0.05% 2mM pH be in 7.4 the borate buffer solution; Promptly accomplish mark, said percentage is mass and size percentage.
6. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 5, said drying is 37 ℃ of oven dryings.
7. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 7, said methyl alcohol is 80% methyl alcohol, and said percentage is percent by volume.
8. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 7, said centrifugal be centrifugal 15 minutes of 2500g.
9. the method for detection fumonisin B1 as claimed in claim 1 is characterized in that, in the said step 7, said dilution is that the 0.05M pH that contains 10% methyl alcohol is 7.4 PBST, and said percentage is percent by volume.
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| CN103421743A (en) * | 2013-05-31 | 2013-12-04 | 华中农业大学 | Recombinant monoclonal antibodies H7 resisting fumonisins B1 |
| CN105709362A (en) * | 2016-04-26 | 2016-06-29 | 中国农业科学院农产品加工研究所 | Application of pseudomonas aeruginosa to fumonisins degradation |
| CN108241059A (en) * | 2016-12-23 | 2018-07-03 | 中粮集团有限公司 | Fumonisin detects colloidal gold quick measuring card and kit and the method being detected to fumonisin |
| CN109085336A (en) * | 2018-08-29 | 2018-12-25 | 郑州工程技术学院 | A kind of immune chromatography test paper detecting fumonisins B1 |
| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
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| CN108241059A (en) * | 2016-12-23 | 2018-07-03 | 中粮集团有限公司 | Fumonisin detects colloidal gold quick measuring card and kit and the method being detected to fumonisin |
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| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
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Application publication date: 20120627 |